Cytosolic phospholipase A2 (cPLA2) has been suggested to be the major isozyme responsible for production of arachidonic acid (AA), the precursor of eicosanoids and to regulate the DNA-binding ability of NFkB. Four different human cPLA2s have been isolated and classified into groups IVA, IVB, IVC, IVD, respectively. Of these isoforms, cPLA2α has been studied most extensively. cPLA2α is an 85 kDa serine esterase, which is found in a wide range of tissues except lymphocytes. By contrast, cPLA2β is a 114 kDa enzyme expressed predominantly in the cerebellum and pancreas, and cPLA2γ is a 61 kDa enzyme expressed predominantly in skeletal muscle. In its inactive state, cPLA2α is located within the cytosol of the cell. cPLA2δ was identified in association with psoriasis. Activation of cPLA2α is regulated by cytoplasmic Ca2+ levels and by phosphorylation, which, in turn, causes its translocation from the cytosol to perinuclear membranes, such as the Golgi, the endoplasmic reticulum and the nuclear envelope. cPLA2α has been shown to be highly selective towards phospholipids that have AA at the sn-2 position. The translocation of cPLA2α is important for at least two reasons: firstly, it enables interaction between the enzyme and its substrate membrane phospholipids, and secondly, it brings the enzyme into close proximity to other downstream enzymes involved in eicosanoid synthesis, specifically COX and LOX.
Although cPLA2α is expressed in several tissue types, its elevated expression has been demonstrated in a range of human tumor types, such as colorectal cancer, small bowel adenocarcinoma, pancreatic adenocarcinoma, esophageal squamous cell carcinoma and lung cancer. Thus, cPLA2α has been postulated to be involved in the pathogenesis of cancer [Laye, J. P. et al. (2003) Drug Discovery Today, 8: 710-6]. Within these tumors, high levels of AA and eicosanoids are observed as a consequence of increased activity of cPLA2α and the COX and LOX enzymes. Like cPLA2α, elevated levels of COX-2 have been associated with human tumourigenesis. Accordingly, selective inhibition of COX-2 activity has attracted considerable interest as an anti-cancer therapeutic strategy. Acetylsalicylic acid (ASA), the most commonly COX-2 inhibitor used, has been shown to reduce the risk for colorectal cancer by as much as approximately 40% In addition, ASA was reported to reduce the risk of colorectal adenoma and carcinoma, as well as experimental colon cancer.
Recently, in human non-small-cell lung cancers, expression of oncogenic forms of Ras were associated with increased expression and activity of cPLA2α—a relationship that was strengthened by the observation that Ras inhibition led to decreased cPLA2α phosphorylation as well as expression, and to prostaglandin synthesis.
Homozygous deletion of the cPLA2α gene in mice resulted in an 83% decrease in small intestinal polyp number and an accompanying decrease in polyp size. The intestinal epithelium in cPLA2α null mice contained numerous small ulcerative lesions, indicating that cPLA2α has a role in tumor promotion, rather than tumor initiation. When compared to wild type, cPLA2α null mice developed 43% fewer urethane induced tumors, indicating a role for cPLA2α in mouse lung tumorigenesis. cPLA2, COX-1, COX-2 and microsomal PGE2 synthase, examined by immunohistochemistry, are present in alveolar and bronchiolar epithelia and in alveolar macrophages in lungs from naïve mice and tumor-bearing mice. Tumors express higher levels of cPLA2, COX-1, COX-2 and microsomal PGE2 synthase when compared to control lungs. Recently, studies have suggested that the effects of cPLA2α on tumor formation might be tissue-specific. Whereas homozygous deletion of cPLA2α produced a significant reduction in tumor number in the murine small intestine, no significant effect was observed in the large intestine [Hong, K. H. et al. (2001). Proc. Natl. Acad. Sci. USA. 98, 3935-3939; Takaku, K. et al. (2000) J. Biol. Chem. 275, 34013-6].
Although the use of COX-2-specific inhibitors, such as ASA, avoids the deleterious side effect of COX-1 inactivation, many of these selective drugs have complications, as a consequence of shifting AA metabolism from the inhibited COX-2 enzyme to alternative pathways, such as COX-1 and LOX and accelerate their activity. Hence, there is a need for alternative therapeutic approaches which would avoid the complications of COX-2 inhibition by limiting AA availability and subsequent eicosanoid production.
Antisense oligonucleotides targeted against the cPLA2 mRNA sequence have been reported in the past as capable of inhibiting cPLA2 transcript expression [U.S. Pat. No. 6,008,344]. However, these oligonucleotides did not demonstrate inhibition of cPLA2 protein expression, and were introduced into cells in the presence of lipofectin.
In addition, three other antisense oligonucleotides targeted to cPLA2 have been described [Roshak, A. (1994) J. Biol. Chern. 269(42): 25999-26005; Muthalif, M. M. et al. (1996) J. Biol. Chem. 271(47): 30149-30157; Marshall, L. (1997) J. Biol. Chem. 272(2): 759-765; Anderson, K. M. et al. (1997) J. Biol. Chem. 272(48): 30504-30511; Li, Q. and Cathcart, M. K. (1997) J. Biol. Chem. 272(4): 2404-2411; Zhao, X. et al. (2002) J. Biol. Chem. 277(28): 25385-25392].
The present inventor designed and described new anti-cPLA2α antisense oligonucleotides, which were more efficient in the inhibition of cPLA2α expression, as well as in the inhibition of pro-inflammatory processes, than the ones previously reported [WO2005/101968]. In the present study, the inventor demonstrates that these antisense oligonucleotides are effective in the inhibition of proliferation of human cancer cell lines.
Thus, it is an object of the present invention to provide the use of anti-cPLA2α antisense oligonucleotides as an anti-neoplastic agent.
Other uses and objects of the invention will become clear as the description proceeds.